Self-assembling peptide surfaces for cell patterning and interactions

ABSTRACT

This invention describes self assembled monolayers (SAMs) manufactured by imprinting reactive peptides onto solid supports. The invention further relates to methods of preparing and using these improved SAMs.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.08/882,415, filed Jun. 25, 1997. The entire teachings of the aboveapplication are incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by a grant(Grant No. 62874) from the Army Research Office. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Organic surfaces have been employed in numerous methods andsystems, including as substrates for ELISA, cell and tissue culture.Self-assembled monolayers (SAMs) are a class of organic surfacesmanufactured by imprinting a monolayer of organic compounds withreactive moieties onto a solid support under conditions wherein thecompounds react with and bind to the solid support in a single orderedand patterned layer. See, Lopez, et al., “Convenient Methods forPatterning the Adhesion of Mammalian Cells to Surfaces UsingSelf-Assembled Monolayers of Alkanethiolates on Gold,” J. Am. Chem.Soc., 115(13):5877-5878 (1993) and Mrksich and Whitesides, “UsingSelf-Assembled Monolayers to Understand the Interactions of Man-MadeSurfaces with Proteins and Cells”, Annu. Rev. Biophys. Biomol. Struct.,25:55-78 (1996). Molecular self-assembly is the spontaneous associationof molecules under equilibrium conditions into stable, structurallywell-defined order joined by non-covalent bonds. SAMs manufactured todate have linked chemical moieties to solid surfaces through long chainalkyl linkages. Examples of organic compounds which have been patternedon a solid support include alkanethiolates and alkylsiloxanes. The SAMsare manufactured employing a process termed “microcontact printing.”

[0004] It has been suggested that SAMs can be used to pattern cells on asurface by presenting chemical moieties which bind to the cells on thesolid surface. Mrksich and Whitesides, above. However, these molecules,and the resulting SAMs, can be difficult and/or expensive tomanufacture. Thus, improvements and cost reductions in the manufactureof SAMs are desirable and are necessary.

SUMMARY OF THE INVENTION

[0005] This invention is based upon the discovery that improved SAMs canbe manufactured by imprinting reactive self assembling peptides ontosolid supports. The SAMs are characterized by ease of manufacture andpurification. They are versatile in their ability to readily provide alarge variety of chemical reactive moieties, or “presenting groups”, toselected targets. For example, the SAM's of the present invention can bereadily designed to present ligands to cellular receptors, cell adhesionmotifs, antibodies or antigen-binding fragments thereof to cell surfaceproteins. This preferred class of SAMs can be used to bind a target,e.g. a selected cell or cells, to a predetermined locus on the solidsupport.

[0006] Thus, the invention relates to a composition of matter comprisinga solid support and a self-assembled monolayer of linear peptideswherein said peptides bound directly to said solid support through aterminal amino acid in a predetermined pattern. Preferably, the peptidescomprise a terminal reactive group, a central linker and a presentinggroup. The invention also relates to the uses and applications of theSAMs described herein, as will be described in more detail below.

[0007] The invention further relates to a method for manufacturing anSAM, or a composition of matter comprising a solid support and aself-assembled monolayer of linear peptides wherein said peptides bounddirectly to said solid support through a terminal amino acid in apredetermined pattern, comprising microcontact printing the reactivepeptides onto the solid support and maintaining the peptides underconditions suitable for binding.

[0008] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings. The drawings are not necessarily to scale,emphasis instead being placed upon illustrating the principals of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIGS. 1 and 2 illustrate methods of microcontact printing reactivepeptides to a solid support in a predetermined pattern.

[0010]FIG. 3 illustrates patterns which may be selected, for example, inSAMs for immobilizing cells.

DETAILED DESCRIPTION OF THE INVENTION

[0011] As set forth above, the invention relates to improved SAMscomprising a predetermined pattern of peptides on a solid support.Preferred peptides of the invention can be characterized by threeregions bound to each other through an amino acid or via peptidebinding, the “terminal reactive group”, the “central linker” and the“presenting group.”

[0012] Upon binding the peptides to the solid support, the peptides arepreferably highly ordered and preferably possess a consistent linear andparallel configuration with each other. Generally, the peptides, or thecentral linker thereof, are fully extended beta strands in configurationunder the conditions of use.

[0013] Although in some embodiments, it may be desirable to present aligand or other molecule which possesses a tertiary structure,generally, the peptides are linear (e.g., free or substantially free ofbranching or tertiary structure). “Substantially free” of branching ortertiary structure is intended to include minor amounts of branching andpeptide interactions which do not significantly interfere with the freemovement or function of the presenting group. The actual degree ofbranching and peptide interactions which can be tolerated withoutdeleteriously effecting the quality of the product will be function ofthe overall length of the peptide, the branched peptides, the nature ofthe amino acids in each and their ability or tendency to interact witheach other can generally be determined by routine screening or computermodeling. For example, peptides “substantially free” of branching mayinclude a peptide composition wherein less than about 5% of the peptidesare characterized by one or more branches.

[0014] While the length of the peptide is not critical to the invention,the peptide is preferably small to moderate in length. Thus, the centrallinker of the peptide can preferably be between about 2 to about 50naturally occurring or non-naturally occurring amino acids in length arepreferred, more preferably between about 8 to about 35 amino acids inlength. Certain peptides in excess of 50 may present undesirableinteractions of the peptides, such as a possible tendency of the peptideto fold. Peptide interactions can be predicted by, for example, computermodeling and structural information available at protein data banks at,for example, Brookhaven National Laboratories, N.Y.

[0015] Peptides which can be used in the invention can be characterizedby a reactive moiety which can react and bind to the solid support, the“terminal reactive group”. Typically, the terminal reactive group is anamino acid characterized by a functional group pendant from the sidechain, the amino group or the carboxy group. Thus, the terminal reactivegroup which binds to the solid support can be an amino acid substitutedby a hydroxy, thiol, carboxy, amino, amido, imido or guanidino group.Preferred terminal amino acids, thus, include serine, cysteine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,arginine and histidine. Alternatively, the terminal reactive group canbe a nonnaturally-occurring amino acid characterized by a functionalitywhich can react with the solid support. Examples include beta aminoacids (amino acids wherein the amino and/or carboxy group are notsubstituted on the same alpha carbon, such as beta-alanine) or aminoacids which have been chemically modified, by electrophilicsubstitution, nucleophilic substitution, activation reactions oraddition reactions, for example. See March, “Advanced Chemistry,” ThirdEdition (1985), Chapters 10-16, the contents of which are incorporatedherein by reference.

[0016] It is further desirable that the peptide be of sufficient lengthto provide a flexible, spatial separation between the solid support(upon reaction with the reactive terminal group) and the opposingreactive terminus of the peptide (e.g., the presenting group). Thus, thepeptides of the invention preferably comprise a “central linker”, whichis a peptide bound to the terminal reactive group and presenting groupthrough peptide or amide bonds. The amino acids employed in the peptideand/or central linker are selected to promote or optimize a beta strandconfiguration at the conditions for use. It is further preferred thatthe amino acids in this portion of the peptide be substantially free oflarge or bulky side chains or bonds which will interfere with theconfiguration (e.g. proline). The amino acids can further be selectedconsidering material strength, permeability and degradation rate of theresulting peptide and SAM. Preferably, the amino acids selected for thecentral section of the peptide are glycine, L-alanine and D-alanine.D-amino acids have advantages in many applications due to theirresistance to L-protease degradation.

[0017] The length of the central linker where present, is also generallynot critical to the invention. Preferably, the central linker is betweenabout 2 and about 30 amino acids in length, more preferably betweenabout 2 and about 8 amino acids.

[0018] The peptide can also be characterized by a “presenting moiety”which will bind to one or more targets. The term “presenting group” isdefined herein to include one or more chemical atoms, functional groups,amino acids or peptides that possess an affinity to or resistance for atarget entity. For example, a presenting group which presents aresistance for a target entity, e.g., a protein or cell, can bepoly(ethylene glycol) or another compound which is inert to the target.A presenting group which is resistant to water, as a target molecule, isa hydrophobic group, such as a high chain alkyl or hydrophobicallyblocked amino acid (e.g., an alkyl ester of valine, leucine, isoleucineor phenylalanine).

[0019] Generally, one or more peptides employed in the present inventionpossess a presenting group with an affinity for a target, e.g. a targetmolecule. In such embodiments, the presenting group can be specific ornon-specific for the target molecule. For example, where the target is acell, the target molecule can be a cell surface protein. The presentinggroup can be a ligand for that protein, an antibody or anantigen-binding fragment thereof which binds specifically to the cellsurface protein.

[0020] The presenting group can be a non-peptide or, preferably, apeptide. As discussed above, the presenting group can be a ligand for oran antibody or antibody fragment which binds to the target molecule.

[0021] Particularly suitable presenting groups are oligopeptides whichself assemble to form a beta sheet under conditions for the desired orselected application. Examples of oligopeptides which self assembleunder these conditions are described in U.S. application Ser. Nos.08/346,849 and 08/784,606, which are incorporated herein by reference intheir entireties. Briefly, these oligopeptides are amphiphilic, havealternating hydrophobic and hydrophilic amino acids and arecomplementary. As will be described in more detail below, particularlypreferred oligopeptides for self assembly are RADX_(n) and EAKX_(n)wherein X is an amino acid and n is an integer between about 2 and about8.

[0022] Particularly preferred targets include cells. Examples of cellswhich can be targeted include prokaryotic and eukaryotic cells. Thecells can be mammalian, plant, bacterial, and yeast. Mammalian cellswhich can be targeted include tumor cells, normal somatic cells and stemcells. The cells can be fibroblasts, endothelial cells, neuronal cells,hepatocytes, blood cells, smooth muscle cells, and progenitors thereof,for example. Bacterial cells can be gram positive or gram negativebacteria and can include Escherichia coli, Streptococcus,Staphylococcus, as well as many others. Bacterial cells which may bedesirable to target and, thus detect and/or culture, can includepathogens and non-pathogens, e.g., contaminants in a food sample, amammalian tissue sample or serum sample or in a plant tissue sample.Similarly, yeast can be targeted and include, for example, Candida andSaccharomyces.

[0023] Cells can preferably be targeted by selecting a presenting groupwhich will react with and bind to the cell surface. Generally, this isaccomplished by binding to a cell surface molecule, such as a protein,lipid, or sugar at the surface of the protein. These surface moleculesare included herein as “target molecules.” For example, a targetmolecule can be a cell surface protein and can be specific to the targetor, in this case, cell, or the target molecule can be non-specific.Where the object of the application is to detect the presence of a cellin a sample, e.g., a tumor cell in a sample which can contain normalcells, it is desirable that the target molecule be specific to the tumorcell (e.g., present on tumor cells and absent on the normal cells).These molecules are generally known in the art as tumor markers. Wherethe object of the invention is to detect the presence of bacteria in asample, such as in food, tissue sample, blood sample, or pharmaceutical,it can be desirable to select a target molecule which is present on manytypes of bacteria which are potentially contaminating the sample to betested. In other instances, e.g., where a substantially pure cellculture is being targeted or transferred to the solid support, theselection of specific or non-specific target molecule is immaterial.

[0024] Suitable target molecules include tumor markers, cellularreceptors, such as CD4 and, CD8. Neuronal cellular receptors includeN-CAMs, the L1 receptors, NGF receptor, the netrin receptor and others.

[0025] Targets can include non-cellular products as well, includingviruses (such as retroviruses, influenza viruses, and herpesviruses, forexample), and proteins (such as prostate soluble antigen (PSA),cytokines, cytokine receptors, growth factors, and growth factorrecpetors. Where the target is a virus, the target molecule can be asurface protein as well, such as a cellular receptor implicated in theinfection of cells. A particularly preferred target molecule for HIV is,for example, gp120.

[0026] Examples of presenting groups include cellular adhesion motifs,ligands or binding fragments of ligands for the target molecule (e.g.,the ligand for gp120 is CD4), antibodies or antigen binding fragments ofantibodies which bind to the target molecule.

[0027] A ligand is defined here to include molecules which are the sameas or substantially the same as the native molecule which binds thetarget molecule. For example, CD4 is a native ligand for the HIV envprotein, gp120. Thus, where the target molecule is gp120, the term“ligand” and, thus, the presenting groups include native CD4, aligand-binding fragment of CD4 (such as, an extracellular domain), andmutations thereof which bind to gp120.

[0028] The terminal reactive group, central linker and presenting groupare preferably arranged linearly with the central linker bonded directlyor indirectly to both the reactive group and the presenting groupthrough, e.g., peptide bonds. Preferably, the peptide has the formula:

X—(CH₂)_(n)—CH(NH₂)CO(AA)_(m)—L

[0029] or

X—(CH₂)_(n)—CH(COOH)NH(AA)_(m)—L

[0030] wherein X is an inert group, such as H, alkyl, alkoxy, alkylthioor dialkylamine, or is a labile or reactive group, such as a thiol,hydroxy, amino, carboxy, acylhalide, carboxy ester, or halide;

[0031] AA is, independently, the same or different, naturally-occurringor non-naturally-occurring amino acid, and is preferably, glycine,L-alanine or D-alanine;

[0032] L is a group which binds specifically or non-specifically to atarget and is preferably a peptide, such as a ligand, an antibody or anantibody fragment;

[0033] n is zero or an integer between 1 to about 5;

[0034] m is an integer of at least about 2 and, preferably, betweenabout 2 and about 50, more preferably between about 2 and about 8.

[0035] The peptides of the invention can be manufactured by known andindustry stadnard peptide synthesis technology. For example, thepeptides can be synthesized chemically or recombinantly (e.g. by theexpression of a recombinant nucleic acid molecule which encodes thepeptide or a precursor thereof). A precursor of the peptide can beparticularly beneficial where one or more of the amino acids arenon-naturally occurring (e.g. a beta amino acid or an amino acid with anon-naturally occurring side chain). The manufacture of peptideschemically and recombinantly are generally practiced in the art and aredescribed in, for example, U.S. application Ser. Nos.: 08/346,849 and08/784,606 and Ausubel, Current Protocols in Molecular Biology (1997).The peptides can preferably be purified prior to use in the manufactureof the SAMs by standard techniques, including HPLC.

[0036] The peptides employed in the invention are imprinted or patternedon a solid support. The shape of the solid support is not critical tothe invention and can be selected to optimize ease of use in theparticular application. Thus, the solid support can be substantiallyspherical (e.g., a bead) or non-spherical, such as in a container (e.g.,a petri dish or cup), cylinder or cone, or a substantially flat film,stick, chip or disc, of essentially any size suitable for the ultimateapplication. The solid support can be porous (as in a membrane) ornon-porous (as in a petri dish or container).

[0037] The material employed in the manufacture of the solid support isnot critical as well. Thus, a variety of materials can be employed inthe manufacture of the solid support. For example, the solid support canbe an inorganic material such as a metal, including as gold, copper,zinc, silver or nickel or a metal alloy. Alternatively, the solidsupport can be glass, silica, or silicon oxide. In yet anotherembodiment, the solid support can be an organic material, such as apolymer or resin, including nylon, poly(ethylene glycol), andpolyfluoropolymers. It can be desirable in some embodiments to employ atransparent solid support. In this embodiment, the detection of thebinding of an opaque target (e.g., a cell) can be determined readily andaccurately visually or electronically and/or robotically employing, forexample, a laser under the control of a computer.

[0038] The solid support is selected with a view towards its ability toreact with the terminal reactive group of the peptide. For example, thethiol group (e.g., X) can react with gold under standard methods, asdescribed, for example in Mrkisch and Whitesides, above. Likewise, thehydroxy group (e.g., X) can react with siloxane under relatively mildconditions. Xia, et al. “Microcontact Printing of Octadecylsiloxane onthe Surface of Silicon Dioxide and Its Application in Microfabrication,”J. Am. Chem. Soc. 117:9576-9577 (1995).

[0039] Solid supports which are inert to the peptide can be derivatizedto render them reactive. For example, the solid support can be coatedwith a reactive material, chemically treated (e.g., by electrophilic ornucleophilic substitution reaction, addition reactions, etc.) tointroduce reactive groups.

[0040] The peptides are printed on the solid support, as will bedescribed below. The terms “printed”, “patterned” or “predeterminedpattern” are defined herein to mean that the solid support has orderedareas where the peptides are bonded and not bonded to the solid support.That is, a printed or patterned solid support is expressly not intendedto include a support with random or substantially homogeneousdistribution of the peptide over its entire surface(s). Furthermore, thepeptides are printed on the solid support in a single layer in asubstantially consistent configuration. Thus, the terms are further notintended to include solid supports wherein peptides are bonded to thesolid support via distinct and different functional groups across thesame molecule (e.g., distinct cysteine residues in a protein containingmultiple cysteines along its sequence).

[0041] The patterns which can be selected in this invention are notparticularly critical. Preferred patterns for SAMs useful as researchtools in the study of cell/cell interactions are linear tracks ofalternating peptides which can adhere to the cells and inert tracks ofsolid support or an inert compound bound to the solid support. Dependingupon the thickness of the tracks, the orientation of the cell canfurther be manipulated. That is a thin track can result in theorientation of the cells linearly. FIG. 3 exemplifies suitable patterns.

[0042] As stated above, methods for the manufacture of SAMs aregenerally known in the art. U.S. Pat. Nos.: 5,620,850 and 5,512,131 andPCT Published Application Nos.: WO97/07429 and WO96/29629 decribedsuitable methods for manufacture. Additional examples include Deng, Li,Milan Mrksich and George M. Whitesides, “Self-Assembled Monolayers ofAlkanethiolates Presenting Tri(propylene sulfoxide) Groups Resist theAdsorption of Protein,” J. Am. Chem. Soc., 118(21):5136-5137 (1996);Chen, Christopher S., Milan Mrksich, Sui Huang, George M. Whitesides,Donald E. Ingber, “Geometric Control of Cell Life and Death,” Science,276:1425-1428 (1997); López, Gabriel P., Mark W. Albers, Stuart L.Schreiber, Reed Carroll, Ernest Peralta, and George M. Whitesides,“Convenient Methods for Patterning the Adhesion of Mammalian Cells toSurfaces Using Self-Assembled Monolayers of Alkanethiolates on Gold,” J.Am. Chem. Soc., 115(13):5877-5878 (1993); Kumar, Amit, Nicholas L.Abbott, Enoch Kim, Hans A. Biebuyck, and George M. Whitesides,“Patterned Self-Assembled Monolayers and Meso-Scale Phenomena,” Acc.Chem. Res., 28(5):219-226 (1995); DiMilla, Paul A., John P. Folkers,Hans A. Biebuyck, Ralph Harter, Gabriel P. López, and George M.Whitesides, “Wetting and Protein Adsorption of Self-Assembled Monolayersof Alkanethiolates Supported on Transparent Films of Gold,” J. Am. Chem.Soc., 116(5):2225-2226 (1994); Singhvi, Rahul, Amit Kumar, Gabriel P.Lopez, Gregory N. Stephanopoulos, Daniel I. C. Wang, George M.Whitesides, Donald E. Ingber, “Engineering Cell Shape and Function,”Science, 264:696-698 (1994); Mrksich, Milan and George M. Whitesides,“Using Self-Assembled Monolayers to Understand the Interactions ofMan-Made Surfaces with Proteins and Cells,” Annu. Rev. Biophys. Biomol.Struct., 25:55-78 (1996); Wilbur, James L., Amit Kumar, Enoch Kim,George M. Whitesides, “Microfabrication by Microcontact Printing ofSelf-Assembled Monolayers,” Adv. Mater. 6(7/8):600-604 (1994); Xia,Younan, Enoch Kim, Milan Mrksich and George M. Whitesides, “MicrocontactPrinting of Alkanethiols on Copper and Its Application inMicrofabrication,” Chem. Mater. 8(3):601-603 (1996); Mrksich, Milan,Jocelyn R. Grunwell and George M. Whitesides, “Biospecific Adsorption ofCarbonic Anhydrase to Self-Assembled Monolayers of Alkanethiolates thatPresent Benzenesulfonamide Groups on Gold,” J. Am. Chem. Soc.,117(48):12009-12010 (1995); Jeon, Noo Li, Ralph G. Nuzzo, Younan Xia,Milan Mrksich, and George M. Whitesides, “Patterned Self-AssembledMonolayers Formed by Microcontact Printing Direct Selective Metalizationby Chemical Vapor Deposition on Planar and Nonplanar Substrates,”Langmuir, 11(8):3024-3026 (1995); Xia, Younan, Milan Mrksich, Enoch Kimand George M. Whitesides, “Microcontact Printing of Octadecylsiloxane onthe Surface of Silicon Dioxide and Its Application in Microfabrication,”J. Am. Chem. Soc., 117(37):9576-9577 (1995). The contents of thesearticles are incorporated herein by reference. The method is illustratedin FIGS. 1 and 2.

[0043] Referring specifically to FIG. 1, a polymeric or elastomericstamp 1 (e.g. a polydimethylsiloxane stamp) is contacted or “inked” witha solution 2 containing the peptide in a suitable solvent and then theinked stamp is pressed against the solid support 3, thereby transferringthe peptide solution in a controlled fashion to the solid support 3. Thepeptide is then maintained in contact with the solid support 3 underconditions suitable for binding, resulting in a SAM 4.

[0044] Upon binding of the peptide to the solid support, the solvent isgenerally removed, for example, by washing (e.g., extraction),evaporation or lyophilization.

[0045] The patterned SAM thus formed can then be used directly or can befurther derivatized, e.g., by subjecting the SAM to a second printingstep to ink a different chemical compound thereon. The second chemicalcompound can preferably be a peptide of the claimed invention or can bedifferent, such as an alkanethiol or poly(ethylene glycol), as describedin Mrksich and Whitesides, above.

[0046] In yet another alternative, the SAM can be subjected toadditional steps which can modify the peptide on the SAM. Thisembodiment may be desirable where the presenting group (e.g., L) or thechemical bond to the central linker ((AA)_(m)) is labile under theconditions for binding the peptide to the solid support. Thus, thepresenting group can be chemically reacted with a peptide precursorbonded directly to the solid support, thereby obtaining a SAM of thepresent invention.

[0047] In many instances, it can be desirable to modify the exposedareas of the solid support, for example, by exposing the SAM toultraviolet light or oxidize the SAM. This can be done to improve thereactivity or eliminate reactivity of the material of the solid supportwith one or more materials encountered in storage or in use of the SAM.

[0048] Referring to FIG. 2, the solid support 3 is stamped with asolution containing a first compound 5 (such as apoly(alkoxyglycothiol)) which can react with the solid support andpresents an imprint or pattern of the solid support, as described above.The printed solid support 6 is then contacted with a solution containingthe peptide 2 under conditions suitable for reacting the peptide withthe exposed solid support. The thus formed SAM 7 possess a pattern ofthe peptide in the relief of the imprint of the first compound. The SAMcan then be washed and dried, as above. The printed solid support 6 canbe immersed into a solution of the peptide or the peptide can be pouredor sprayed onto the surface of the SAM, as is convenient.

[0049] Solvents which can be used to ink the peptide onto the stamp and,then, onto the solid support include solvents which can disperse or,preferably, solubilize the peptide. The solvent is preferably readilyremoved, for example, by evaporation, lyophilization or extraction, fromthe solid support. Examples of preferred solvents include alcohols, suchas ethanol, acetone, acetonitrile, DMSO and DMF and misciblecombinations thereof. The peptide solution concentration is selectedsuch that the desired amount of peptide is delivered to the solidsupport. That is, if it is desired to print the peptide upon the solidsupport at a high concentration or density, then the peptide solutioncan be at or near the saturation level of a good solvent. If it isdesired to imprint a low concentration of the peptide sparsely upon thesolid support, the solution can be characterized by a low concentrationsuch as employing a dilute solution.

[0050] The solution comprising the peptide can also include additionalcomponents. For example, a dispersant or solubilizer can be added to thesolution to solubilize or disperse, for example, the peptide. It can bedesirable in some instances to include a colorant in the solution,particularly where the solution is colorless or is difficult to observeon the solid support or stamp, so that the area of the solid supportwhich has been inked can be visually observed. It is generally desirablewhere additional components are added to the solution that they can bereadily removed from, e.g. washed free of, the solid support.

[0051] It is clear that, in the method for manufacturing the SAMs,either the stamp, solid support or both can be mobile, relative to theother. That is, the stamp can be fixed and the solid support pressedfirmly against it or vice versa. Alternatively, both the stamp andsupport can be mobilized. This process can be readily achieved employingrobotics, which ensures a high degree of consistency and accuracy in theprinting step.

[0052] The peptide can be bound to the solid support via covalentbonding, ionic bonding or other chemical interactions. It is preferredthat the bonding be of a high affinity and be essentially irreversibleunder the conditions for use. The conditions suitable for bonding thepeptide to the solid support can be dependent upon the nature of thechemical reaction relied upon and can generally be determined by theperson of skill employing no more than routine skill.

[0053] Clearly, other methods for the manufacture of the SAMs of thepresent invention will be apparent to the person of skill in the art andare intended to be included within the scope of the present invention.

[0054] The SAMs of the invention can be employed in a variety ofprocesses in biology, biotechnology, medicine, material science,biomedical engineering and computer-related inventions. A preferredexample of an application includes the use of the SAMs as substrates forELISA.

[0055] SAMs to Screen for the Presence of a Target in a Sample

[0056] The SAMs of the present invention can be used to screen for thepresence of a target in a sample. As set forth above, the SAMs of theinvention can be designed to possess a presenting group which bindsspecifically or non-specifically to a target or target molecule. Wherethe presence of a cell is to be detected and distinguished from othercells in the sample (e.g.,the presence of a tumor cell in a tissuesample which can further comprise normal diploid cells), the presentinggroup is “specific” to the target, i.e. does not bind substantially toother materials or cells which can be present. Where the presence ofmany different cells in a sample (e.g., the presence of bacterialcontaminants in a pharmaceutical process stream), the presenting groupis non-specific to a particular target but can bind to a large number oftargets.

[0057] The method of screening for the presence of a target can comprisethe steps of contacting an SAM, as described above, with a sample underconditions suitable for the target or target molecule to bind to thepresenting group on the SAM and detecting the presence of the target ortarget molecule. The target or target molecule can be a cell, such as amammalian cell (e.g., tumor cell, normal diploid somatic cell, or stemcell), a bacterium or yeast (e.g., a causative agent for disease orcontaminant). Alternatively, the target or target molecule can be avirus (e.g., a causative agent for disease or contaminant), toxin orprotein, etc.

[0058] The sample can be obtained from an animal or patient, such as atissue sample or biopsy, body fluid, e.g., serum, milk, saliva or urineor fecal matter. Alternatively, the sample can be obtained frommanufacturing process, such as a pharmaceutical process or food process.Thus, the method can be used to screen for contaminants or sterileconditions in manufacturing or it can be used to screen for or diagnosedisease in a patient.

[0059] It is generally desirable that the sample be contacted with theSAM as a liquid, e.g. a dispersion or solution. Thus, the sample can bemixed with a diluent or buffer. Examples of diluents include water, suchas sterile water, polar and non-polar solvents, e.g. alcohols,dimethylformamide, acetonitrile, alkanes, benzene, toluene, etc. Buffersinclude physiological buffers, such as phosphate buffered solution,culture media, etc.

[0060] The person of skill in the art can determine empirically theconditions for contacting the SAM and the sample such that the target ortarget molecule can react with each other and bind. Such conditions arewell known in the art. Generally, where the method is a diagnostic tooland the sample is a tissue sample or other biological sample, theconditions will physiologic. That is, physiological pH is generallyemployed. Room temperature can also be employed in many instances. Wherethe method is detecting the presence of contaminants in a sample,neutral pH can be generally employed, as well as room temperature.

[0061] The SAM can be contacted with the sample in a number of ways. Forexample, the SAM can be immersed into the sample, as in dipping a stick.Alternatively, the sample can be poured over or through the SAM.Optionally, the SAM can be rinsed after the contacting step, such aswith sterile water.

[0062] After the SAM has been contacted with the sample, the presence ofthe target or target molecule is detected. This can also be performed ina number of ways. In one embodiment, the SAM can be contacted with asecond solution which possesses a labeled compound which can react withthe target molecule, as in an ELISA method. The label (e.g., acolorimetric label or radiolabel) can then be detected. In manyembodiments, the target can be detected visually, with the naked eye,under a microscope or robotically. This can be advantageous, forexample, where the target is a cell. In many embodiments, it may bedesirable to permit any cells bound to the SAM to colonize prior todetection. The method of the invention can accurately determine thepresence of an individual cell or determine a precise cell count in asample.

[0063] In a particularly preferred method, the solid support for the SAMis transparent. In such an embodiment, the presence of an opaque target,such as a cell, can be determined by scanning the SAM with a laser anddetermining the number of targets or cells present thereon, whichaccurately correlates to the number of interruptions in scanning. Thismethod can be performed in an automated system (e.g. robotically),thereby improving efficiency and avoiding inaccurate results due tohuman error.

[0064] SAMs in Cell Culture

[0065] The SAMs of the invention can be used as a solid support inculturing cells. Cells can be attached to the SAMs by contacting thecells to be attached with the SAM and maintaining the cells underconditions suitable for growth. As above, it is generally desirable tocontact the cells with the SAM as a liquid, e.g., in the presence of adiluent or solvent. The cells can be attached to the solid support in apredetermined fashion, order and orientation.

[0066] Conditions for maintaining cells can be those employed routinelyfor the cell or cell type to be cultures. For example, the culture canbe maintained under temperatures (e.g. between about 25° C. to about 60°C.) and pH (e.g. between about 4 and about 10) appropriate for growth.Nutrients appropriate for growth can also advantageously be provided tothe culture.

[0067] The invention permits very accurate control of cell populationand density. The invention can be utilized to study cell growth andcellular interactions to external stimuli, including other cells, growthfactors, repellants and inhibitors. Thus, the invention represents asignificant advance in the ability to conduct research in biology andmedicine.

[0068] In yet another embodiment, the method can be employed inscreenings or assays employing cells, such as screening for drugs whichmay inhibit the growth of a cell or cells (such as in a screen foranti-tumor agents, anti-bacterials). Alternatively, the method can beemployed in the screening for drugs which increase or activate thegrowth of a cell or cells, including fibroblasts, endothelial cells,smooth muscle cells, hematopoietic cells and neuronal cells, etc.

[0069] The method can also be used to maintain cell cultures, includingtissue cultures, in the manufacture of cellular products (e.g.,proteins, hormones, etc.), artificial tissues, etc. Examples of tissueswhich can be cultured in this manner include fibroblasts, endothelialcells, smooth muscle cells and neuronal cells. Such tissues can beemployed as grafts, such as autologous grafts.

[0070] The understanding of complex neuronal connections is central toour comprehension of central nervous system function, and advances indoing so will benefit from combining engineering with molecular cellbiology to analyze neuronal behavior under well-characterized andcontrolled conditions. Neurite outgrowth, guidance and connections canbe studied on surfaces patterned with self-assembling peptides thatcontain cell-adhesion motifs. Controlling neurite outgrowth, includingdistances, angles and direction, can be important in controlling andstudying synapse formation between neuronal cells guided into proximity.Neuronal cells attached to the described SAMs can be employed in thestudy of neuronal cell culture, synapse formation, neuronal connectionengineering, screening neuropeptides, as well as pharmaceutical agentsthat stimulate, inhibit or alter the nature of nerve growth, andinter-connections. For example, attractants, e.g., growth factors,neuropeptides, neurotrophins, and drugs can be screened for theirability to alter the direction or growth behavior of neurites or theirability to induce, stimulate, suppress or inhibit neurite growth. Theseattractants can be placed or randomly contacted with the neuronalcell-bound SAMs.

[0071] Preferred peptides for the manufacture of the SAMs for thisapplication include peptides wherein the presenting group is a celladhesion motif or peptide which binds to neuronal cells. Examples ofsuitable cell adhesion motifs are (RADX)_(n), (RADS)_(n), (EAKX)_(n),and (EAKS)_(n), wherein X is an amino acid, such as S, and n is aninteger, preferably between about 2 to about 8. Oligopeptides of thesesequences have been shown to promote neurite outgrowth in culture (U.S.application Ser. No. 08/784,606, which is incorporated herein byreference in its entirety).

EXAMPLE 1

[0072] Preparation of Patterned SAMs Glass Chip

[0073] A 10:1 (w:w) mixture of Sylgard Silicone Elastomer 184 andSylgard Curing Agent 184 (Dow Corning Corp., Midland, Mich.) was castedover a master, which was generated by photolithography, and pressuredegassed. After sitting at room temperature for 1 hour, the PDMS wascured at 60° C. for 2 hours. The stamp was carefully peeled off themaster after cooling to room temperature and rinsed with ethanol. ThePDMS stamp was inked by a cotton swab which has been moistened with a 5mM solution of (1-mercaptoundec-11-yl)hexa(ethylene glycol)(HO(CH₂CH₂O)₆(CH₂)₁₁SH) in ethanol. The resulting stamp was placed onthe gold substrate (125 Å gold on a titanium-primed 24×50-2 microscopecover glass) and gentle hand pressure was applied to aid in completecontact between the stamp and the glass chip. After 1 minute, the stampwas peeled off the glass chip and the resulting substrate was immerseddirectly in a 2 mM solution of (RADC)₃ AAAC (SEQ ID NO: 1) in distilled,deionized water. After approximately 2 hours of immesion, the glass chipwas removed from the solution, rinsed extensively with water andethanol, and dried with a stream of filtered nitrogen gas.

[0074] In our preliminary experiments, when the cells (of various types)are plated on surfaces coated with hexa-ethyleneglycolthiol, (EG)6-SH,they rarely attach to the surface. In contast, cells attached very wellwhen plated on the surface coated with the “RADSC” peptide. In theseexperiments, after cell attachment, the plates containing cells weeshacked at 150 rpm for 10 minutes and the coated cover-slides werewashed in new medium and transferred to new plates in order to eliminateunattached cells.

[0075] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A composition of matter comprising a solidsupport and a self-assembled monolayer of linear peptides bound in apre-determined pattern to said solid support by a bond between the solidsupport and a terminal amino acid of the linear peptides, wherein saidlinear peptides comprise: a) a presenting group which binds specificallyto a cell surface protein; and b) a central linker between thepresenting group and the terminal amino acid.
 2. The composition ofmatter according to claim 1 wherein said solid support is a metal. 3.The composition of matter according to claim 2 wherein said metal isselected from the group consisting of gold, copper, nickel, zinc andsilver.
 4. The composition of matter according to claim 1 wherein saidsolid support is selected from the group consisting of silica and glass.5. The composition of matter according to claim 1 wherein the terminalamino acid is selected from the group consisting of serine, cysteine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,histidine and arginine.
 6. The composition of matter according to claim5 wherein said terminal amino acid is selected from the group consistingof serine, aspartic acid, glutamic acid and cysteine.
 7. The compositionof matter according to claim 6 wherein said central linker comprisesbetween 2 to 50 amino acids.
 8. The composition of matter according toclaim 7 wherein said central linker is selected from the groupconsisting of a oligoglycine and oligoalanine.
 9. A composition ofmatter comprising a solid support and a self-assembled monolayer oflinear peptides bound in a pre-determined pattern to said solid supportby a bond between the solid support and a terminal amino acid of linearpeptides.
 10. The composition of matter according to claim 9 whereinsaid solid support is a metal.
 11. The composition of matter accordingto claim 10 wherein said metal is selected from the group consisting ofgold, copper, nickel, zinc and silver.
 12. The composition of matteraccording to claim 9 wherein said solid support is selected from thegroup consisting of silica and glass.
 13. The composition of claim 9wherein the linear peptides comprise a presenting group selected fromthe group consisting of a peptidyl group, antigen, antibody, antibodyfragment, cellular adhesion motif, high chain alkyl, hydrophobicallyblocked amino acid and ligand and a central linker between the terminalamino acid and the presenting group.
 14. The composition of claim 13wherein the presenting group is a peptidyl group which possesses anaffinity to a target molecule.
 15. The composition of matter accordingto claim 13 wherein the terminal amino acid is selected from the groupconsisting of serine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, histidine and arginine.
 16. Thecomposition of matter according to claim 15 wherein said terminal aminoacid is selected from the group consisting of serine, aspartic acid,glutamic acid and cysteine.
 17. A method for manufacturing a compositionof matter comprising a solid support and a self-assembled monolayer oflinear peptides bound in a pre-determined pattern to said solid support,said method comprising the steps: (a) contacting an elastomeric stampcharacterized by a relief of said predetermined pattern with a solutioncontaining a compound which can react with said solid support; (b)contacting said stamp with a surface of said solid support underconditions suitable for the reaction between said compound and saidsolid surface, wherein said compound reacts with said solid support atpoints of contact between said stamp and said solid support,corresponding to the relief of said predetermined pattern; (c) removingsaid stamp; and (d) contacting said solid support with a solutioncontaining said linear peptides under conditions suitable for thereaction of said peptide and said solid support.
 18. A method forculturing cells comprising the steps of: (a) contacting the compositionof matter of claim 1 with cells having the cell surface protein to whichthe presenting group specifically binds under conditions suitable forsaid cells to bind to the presenting group; and (b) maintaining saidcells under conditions suitable for growth.
 19. A method for culturingcells comprising the steps of: (a) contacting the composition of matterof claim 9 with cells under conditions suitable for said cells to bindto the peptides; and (b) maintaining said cells under conditionssuitable for growth.
 20. A method for assaying the presence of a targetin a sample comprising the steps of: (a) contacting said sample with thecomposition of matter of claim 9 said linear peptides possess anaffinity for said target; and (b) detecting the presence of said targeton said composition of matter.